The pharmaceutical biotechnology industry is based on the production of recombinant proteins in mammalian cells. These proteins are essential to the therapeutic treatment of many diseases and conditions. In particular, antibodies are of increasing importance in human therapy, assay procedures and diagnostic methods. However, methods of identifying antibodies and production of antibodies is often expensive, particularly where monoclonal antibodies are required. Hybridoma technology has traditionally been employed to produce monoclonal antibodies, but these methods are time-consuming and result in isolation and production of limited numbers of specific antibodies. Additionally, relatively small amounts of antibody are produced; consequently, hybridoma methods have not been developed for a large number of antibodies. This is unfortunate as the potential repertoire of immunoglobulins produced in an immunized animal is quite high, on the order of >1010, yet hybridoma technology is too complicated and time consuming to adequately screen and develop large number of useful antibodies.
One approach to this problem has been the development of library screening methods for the isolation of antibodies (Huse et al., Science 246:1275 [1989]; McCafferty et al., Nature 348:552 [1990]). Functional antibody fragments have been produced in E. coli cells (Better et al., Science 240:1041 [1988]; Sastry et al., PNAS 86:5728 [1989]) as “libraries” of recombinant immunoglobulins containing both heavy and light variable domains (Huse et al., supra). The expressed proteins have antigen-binding affinity comparable to the corresponding natural antibodies. However, it is difficult to isolate high binding populations of antibodies from such libraries and where bacterial cells are used to express specific antibodies, isolation and purification procedures are usually complex and time-consuming.
Combinatorial antibody libraries generated from phage lambda (Huse et al., supra) typically include millions of genes of different antibodies but require complex procedures to screen the library for a selected clone. Methods have been reported for the production of human antibodies using the combinatorial library approach in filamentous bacteriophage. A major disadvantage of such methods is the need to rely on initial isolation of the antibody DNA from peripheral human blood to prepare the library. Moreover, the generation of human antibodies to toxic compounds is not feasible owing to risks involved in immunizing a human with these compounds.
Currently the most widely used approach for screening polypeptide libraries is to display polypeptides on the surface of filamentous bacteriophage. The polypeptides are expressed as fusions to the N-terminus of a coat protein. As the phage assembles, the fusion proteins are incorporated in the viral coat so that the polypeptides become displayed on the bacteriophage surface. Each polypeptide produced is displayed on the surface of one or more of the bacteriophage particles and subsequently tested for specific ligand interactions. While this approach appears attractive, there are numerous problems, including difficulties of enriching positive clones from phage libraries. Enrichment procedures are based on selective binding and elution onto a solid surface such as an immobilized receptor. Unfortunately, avidity effects arise due to multivalent binding of the phage and the general tendency of phage to contain two or more copies of the displayed polypeptide. The binding to the receptor surface therefore does not depend solely on the strength of interaction between the receptor and the displayed polypeptide. This causes difficulties in the identification of clones with high affinity for the receptor.
Thus, the art is in need of efficient methods of generating and screening antibody libraries containing large numbers of antibodies.